Hepatitis C virus infection induces autocrine interferon signaling by human liver endothelial cells and release of exosomes, which inhibits viral replication

Abstract

Background & aims: Liver sinusoidal endothelial cells (LSECs) make up a large proportion of the nonparenchymal cells in the liver. LSECs are involved in induction of immune tolerance, but little is known about their functions during hepatitis C virus (HCV) infection.

Methods: Primary human LSECs (HLSECs) and immortalized liver endothelial cells (TMNK-1) were exposed to various forms of HCV, including full-length transmitted/founder virus, sucrose-purified Japanese fulminant hepatitis-1 (JFH-1), a virus encoding a luciferase reporter, and the HCV-specific pathogen-associated molecular pattern molecules. Cells were analyzed by confocal immunofluorescence, immunohistochemical, and polymerase chain reaction assays.

Results: HLSECs internalized HCV, independent of cell-cell contacts; HCV RNA was translated but not replicated. Through pattern recognition receptors (Toll-like receptor 7 and retinoic acid-inducible gene 1), HCV RNA induced consistent and broad transcription of multiple interferons (IFNs); supernatants from primary HLSECs transfected with HCV-specific pathogen-associated molecular pattern molecules increased induction of IFNs and IFN-stimulated genes in HLSECs. Recombinant type I and type III IFNs strongly up-regulated HLSEC transcription of IFN λ3 (IFNL3) and viperin (RSAD2), which inhibit replication of HCV. Compared with CD8(+) T cells, HLSECs suppressed HCV replication within Huh7.5.1 cells, also inducing IFN-stimulated genes in co-culture. Conditioned media from IFN-stimulated HLSECs induced expression of antiviral genes by uninfected primary human hepatocytes. Exosomes, derived from HLSECs after stimulation with either type I or type III IFNs, controlled HCV replication in a dose-dependent manner.

Conclusions: Cultured HLSECs produce factors that mediate immunity against HCV. HLSECs induce self-amplifying IFN-mediated responses and release of exosomes with antiviral activity.

Keywords: Cytokine; Immune Regulation; Innate Immunity; Toll-Like Receptor.

Figure 1. Characterization of primary HLSECs

FACS analysis demonstrates that primary HLSECs are negative for the pan-leukocyte antigen CD45 but do express classic endothelial-associated antigens (A). Several receptors, involved in HCV binding and entry, are also expressed (B). HLSECs are positive for certain Toll-like receptors (TLRs) and RIG-I (further induced by IFN-α stimulation Western blotting) (C). Shaded histograms represent isotype controls.

Figure 2. HCV is taken up by HLECs

Primary HLSECs (A), TMNK-1 (B) and Huh7.5.1 cells (C) were plated for 24 hours and stained for Keratin 18 (K18, green) and F-actin (red). Endothelial cells contrary to hepatocytes do not express K18 (magnification 40X). Co-culture experiment with primary HLECs and Huh7.5.1 was performed as described in the Methods. Cells were stained for NS5A (blue), F-actin (red), K18 (green) and DAPI. HCV-NS5A is detected in both cell types, confirming that HLSECs can uptake HCV (magnification 40X) (D). Primary HLSECs were plated for 24 hours with supernatant of JFH-1-infected Huh7.5.1. Cells were stained for F-actin (red) and core or NS5A (blue) (magnification 40X) (E). The vast majority of TMNK-1 cells exposed to cell-free supernatant from JFH-1-infected Huh7.5.1 cells demonstrated HCV-NS5A protein (F) (magnification 25X). Cells were visualized using confocal microscopy as described in the Methods. Bar represents 10 μm.

Figure 3. HLSECs respond to various forms of HCV full-length virus

Full-length genotype 1a HCV T/F vRNA was added exogenously to TMNK-1 cells for 24 hours (normalized to mock) in presence (white bars) or absence of (black bars) chloroquine (100 μM) (n=3). Chloroquine significantly inhibits up-regulation of Type I/III IFN genes in response to exogenous T/F vRNA. Mann-Whitney test, two-tailed (A). TMNK-1 cells were treated with sucrose-purified JFH-1 virus for 48 hours at MOI=0.08 (n=3, normalized to non-infected). Wilcoxon Signed rank Test (B). Full length JFH-1-RNA was transfected into TMNK-1 cells for 24 hours (normalized to non-infected) (n=5). Wilcoxon signed-rank test (C). Full length JFH-1-RNA was transfected into TMNK-1 cells for 24 hours in the presence of the IRS661 (TLR7 specific antagonist) or the control (IRS-Ctrl) (n=3). Fold increase of the mRNA was normalized to the IRS-Ctrl considered 100%. IFNA1 and IFNA2 were not included because they were not up-regulated by transfected JFH-1. Wilcoxon signed-rank test (D). For every experiment gene up-regulation was assessed by real-time RT-PCR. Bars represent mean plus SEM, * p<0.05, ** p<0.01, ***p< 0.001, ****p< 0.0001.

Figure 4. HLSECs sense HCV-RNA and demonstrate differential response to TLR3 and TLR4 ligation

Primary HLSECs were transfected with the HCV-PAMP for 8 hours (normalized to X-region control) (n=8) (A). Protein secretion was higher in supernatants of HCV-PAMP-transfected primary HLSECs compared to control as assessed by ELISA (representing 3 to 7 experiments) (B). Primary HLSECs stimulated with Poly I:C (TLR3) (C) or LPS (TLR4) (D) (normalized to mock) showed different transcriptional profiles (n=4). Gene up-regulation was assessed by real-time RT-PCR. Bars represent mean plus SEM, Wilcoxon signed-rank test, * p<0.05, ** p<0.01, ****p< 0.0001.

Figure 5. IFN stimulation of HLSECs induces IFN and ISG responses

Primary HLSECs were stimulated with pegylated-IFN-α2 (n=3) or Type III IFNs (n=4) for 8 hours. Pegylated-IFN-α2, IFNL2, IFNL3 and IFNL1 stimulation induced significant up-regulation of IFNL3 (normalized to mock) (A). Primary HLSECs stimulated with pegylated-IFN-α2 (n=3) for 8 hours induced up-regulation of multiple ISGs (B) not observed in primary HLSECs simulated with Type III IFNs (n=4) (C). Stimulation with pegylated-IFN-α2 for 24 hours (n=3) up-regulated ISGs, particularly RSAD2 (confirmed by Western blot analysis as shown in the inset) (D). All of the Type III IFNs induced significant up-regulation of RSAD2 after 24 hours stimulation (E). Gene up-regulation was assessed by real-time RT-PCR. Bars represent mean plus SEM, Wilcoxon signed-rank test, * p<0.05, ** p<0.01, ***p< 0.001.

Figure 6. HLSECs demonstrate increased antiviral potential compared to non-liver vascular ECs, up-regulate antiviral genes in HCV-infected livers, and control HCV replication in vitro

Pathway analysis demonstrating over-representation of Type I (IFNA2, activation z-score 4.459, p-value=2.89×10⁻¹⁵) and Type III (IFNL1, activation z-score 3.973, p-value=4.30×10⁻¹¹) interferon upstream regulators in TMNK-1 cells (A). Quantitative PCR confirmed relevant ISGs (B), fold changes relative to mock HMEC. Wilcoxon sign-ranked test used to determine significance (**p<0.005). On day 4, CD8⁺ T (white bar) or TMNK-1 cells (black bar), were added to infected Huh7.5.1 for 24 hours (n=3). HCV viral copy number (C), and gene up-regulation (D) were assessed as described in the Methods. Results were normalized to CD8⁺ T cells/Huh7.5.1 co-culture considered 100%. Mann-Whitney test, two-tailed. Liver sinusoidal endothelial cells (CD45-CD31+) isolated form HCV-infected individuals significantly up-regulate IFNB, ISG15 and OAS1 compared to healthy controls (n=3) (E). Exosomes derived from mock or IFN-treated TMNK-1 cells were added to Huh7.5.1 at the time of infection in increasing doses (5-10-25 μg/ml) for 5 days (F). Exosomes from Peg-IFNα2-treated TMNK-1 were isolated after 24 hours (n=1) and 48 hours (n=1) with pooled data displayed. Exosomes from IFN-λ1/2/3-treated TMNK were isolated after 48 hours (n=3). Results were normalized to Huh7.5.1/mock-treated exosomes culture considered 100%. One-sample t test. Bars represent mean plus SEM, *p<0.05** p<0.01, ***p< 0.001, ****p< 0.0001.

Figure 7. Paradigm for roles of HLSECs in HCV entry, uptake, recognition, and replication

HCV is pinocytosed and viral RNA is translated within HLSECs. HLSECs respond with production of Type I/III IFNs and ISGs activation. This enhanced antiviral state contributes to prevention of new infection within HLSECs, i.e., “viral repulsion” and inhibition of viral replication within hepatocytes. HLSECs supernatants, in addition to stimulating STAT1 induce up-regulation of SOCS1 that may counter-regulate the antiviral response in hepatocytes. Exosomes produced by HLSECs following stimulation with either Type I/III IFNs have antiviral properties.